Nitrosation-inducible inhibitors biological macromolecules

ABSTRACT

Biomacromolecules such as proteins are inactivated by hydrophobic ANSA derivatives of the formula:  
                 
 
     wherein R 1  and R 2  are hydrophobic or affinity groups and R 3  is selected from the group consisting of aminoacyl groups and peptidyl groups upon nitrosation. ANSA derivatives can be designed to selectively kill tumor cells and various pathogens, including bacteria, viruses, and fungi.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of Ser. No. 09/988,397,filed Nov. 19, 2001, which claims priority from provisional applicationSerial No. 60/249,277, filed Nov. 17, 2000, the entire contents of whichare hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for inactivatingbiomacromolecules in order to regulate their metabolism. This method isparticularly useful in combating pathogens and cancer cells.

BACKGROUND OF THE INVENTION

[0003] Nitric oxide (*NO) is an essential bioactive molecule thatmediates a variety of actions, such as vasodilation, neurotransmission,and host defense. However, increased *NO production is often associatedwith the pathogenesis of various disorders, including cancer (Moncada etal, 1991; Kerwin et al, 1995). *NO is produced endogenously by a familyof enzymes known as *NO synthases (NOS). Only the inducible isoform(iNOS) produces *NO concentrations in the micromolar range (Nathan,1997), which is high when compared with the pico- to nanomolarconcentrations produced by neuronal (nNOS) and endothelial (eNOS)isoforms (Brovkovych et al, 1999). iNOS is highly active in inducedmacrophages during chronic and acute inflammation causing excessivenitrosation (covalent attachment of nitroso group to thiols or amines)of proteins, lipids and nucleic acids—a condition known as nitrosativestress.

[0004] Recent studies indicate that the increases NOS expression andactivity may contribute to tumor development or progression. High levelsof *NO synthesis were observed in human gynecological (Thomsen et al,1994), prostate (Klotz et al, 1998), breast (Thomsen et al, 1995), colon(Ambs et al, 1998), and central nervous system tumors (Cobbs et al,1995). High levels of *NO are also observed in the most common chronicinflammatory diseases of digestive tract, which predispose individualsto cancer (Wilson et al, 1998; Mannick et al, 1996; Al-Mufti et al,1998). Furthermore, most infectious bacteria produce a significantamount of NO endogenously and also become a subject to amacrophage-derived NO.

[0005] *NO is chemically unreactive toward most bioorganic compounds,but it spontaneously auotoxidizes to yield the highly reactive species,N₂O₃—the actual nitrosating agent (Williams, 1997; Kharitonov et al,1995; Grisham et al, 1999) (FIG. 1A). Nitrosation of biologicalsubstrates occurs with high efficiency during nitrosative stress in bodyfluids and tissues (Grisham et al, 1999; Ischiropoulos, 1998). Toexplain the mechanism of nitrosation in vivo and particularly undernoninflammatory conditions when the concentration of free NO is low, themechanism of micellar catalysis of NO oxidation has been put forward(Liu et al, 1998; Nedosapasov et al, 2000). NO and O₂ are bothhydrophobic molecules, areas of high hydrophobicity can act as a“sponge” to sequester them from the surrounding aqueous phase. Highlocal concentrations of NO and O₂ in a hydrophobic phase, e.g. withinlipid membranes, can significantly accelerate NO oxidation and N₂O₃formation (Liu et al, 1998; Nedosapasov et al, 2000). Recently, wedemonstrated that such micellar catalysis of NO oxidation occurs withinthe hydrophobic cores of various soluble proteins (Nedosapasov et al,2000) (FIG. 1B). For example, serum albumin can accelerate the formationof N₂O₃ more than 15,000 times (Rafikova et al, 2001).

[0006]FIG. 1 shows micellar catalysis of *NO oxidation and nitrosationof biological substrates. FIG. 1 shows the third order reaction of *NOwith O₂ (k=6×10⁶ M⁻² sec⁻²) (Wink et al, 1994). FIG. 1 shows hydrophobiccompartments (micelles) formed by a protein globule accumulate *NO andO₂ from aqueous solution thus accelerating the formation of reactivenitrosating species, N₂O₃. N₂O₃ can react with water only at the surfaceof the protein (via intramolecular NO⁺ transfer) to form nitrite (NO₂⁻). At the same time, various nucleophiles (e.g. thiols [SH]) canpenetrate the protein interior or exist already inside and be accessiblefor nitrosation (Nedosapasov et al, 2000).

SUMMARY OF THE INVENTION

[0007] It is an object of the present invention to overcome deficienciesin the prior art.

[0008] It is another object of the present invention to provideselective therapy to treat infections and cancer using ANSA derivatives.

[0009] It is a further object of the present invention to provide amethod to inactivate a broad range of pathogens such as bacteria, fungi,viruses, by effecting nitrosation within the pathogen.

[0010] According to the present invention, specific hydrophobiccompounds are provided which cooperate with *NO and oxygen to rapidlyinactivate target proteins. These compounds are preferablyaminonaphthalenesulfonamide (ANSA) derivatives which are precursors ofcompounds which are generated in vivo to inactivate proteins. Thesederivatives are hydrophobic and contain a peptidyl moiety which targetsthe derivative to a specific cell.

[0011] Formation of nitrosating N₂O₃ (NO⁺—NO₂ ⁻) within proteininteriors does not necessarily lead to permanent protein nitrosation anddamage, since the nitrosonium cation (NO⁺) is readily transferred out ofthe protein globule into H₂O to form relatively benign nitrite, as shownin FIG. 1B. However, with the help of additional small chemicals (ANSA),this process can be directed towards rapid protein inactivation.

[0012] The compounds of the present invention have three variablemoieties chemically attached to ANSA, as shown below:

[0013] wherein at least one of R₁ and R₂ is a group which impartshydrophobicity to the molecule and R₃ is either aminoacyl or peptidyl.When R₃ is an aminoacyl group, the aminoacyl group generally has fromabout 1 to about 30 carbon atoms. Either R₁ or R₂ can be an affinitygroup for, e.g., a virus, such as a monoclonal antibody. When R₁ or R₂is an affinity group, the other of R₁ or R₂ is selected such that themolecule is hydrophobic.

[0014] Groups which import hydrophobicity to the molecule, i.e.,hydrophobic groups, include non-polar groups, such as C₈-C₃₀ alkyl,alkenyl, alkynyl, arylalkyl, and alkylaryl groups. One skilled in theart can readily determine what groups would impart hydrophobicity to themolecule.

[0015] The rate of nitric oxide-dependent nitrosation of hydrophobicaromatic amines in the presence of oxygen is increased in heterogenicmedia by micellar catalysis. The product of this reaction, aryldiazoniumion, is highly reactive, and rapidly interacts with biomacromolecules toinactivate these molecules. ANSA is superior to conventional hydrophobicaromatic amines because:

[0016] (1) ANSA can readily be manipulated to change the hydrophobicproperties;

[0017] (2) conventional hydrophobic aromatic amines (e.g., aniline,naphthylamines) accumulate in mammalian organs (especially the liver)and form carcinogenic products upon oxidation. The presence of thesulfonamide group in ANSA prevents these compounds from accumulatingbecause they are enzymatically hydrolyzed to sulfonic acid;

[0018] (3) coupling between amino and sulfonamide groups allows theformation of highly reactive aryldiazonium products;

[0019] (4) straightforward procedures for synthesizing ANSA and theirderivatives have already been developed, particularly as described inUSSR patents 1586902, 1814808 and 1648054.

[0020] As shown in FIG. 2A, nitrosation of the amino group converts thefluorescent ANSA into the aryl-diazonium cation that crosslinks toanother ANSA molecule in solution or in the protein interior and losesits fluorescent activity. Coupling of two or more ANSA moleculesproduces non-fluorescent dyes. In FIG. 2 ANSA, R₁, R₂, and R₃ stand forvariable substitutions. If R₃ is substituted the ANSA is unable toperform azocoupling reactions.

[0021]FIG. 2B shows the mechanism of ANSA-dependent proteininactivation. The hydrophobic ANSA molecules with an intact NH₂ group(ANH₂) accumulate inside the protein hydrophobic core and form reactivediazonium cations (AN₂ ⁺) upon nitrosation. AN₂ ⁺ crosslinks with nearbyfunctional amino acids and inactivates the protein.

[0022]FIG. 2B illustrates the scenario. First, a protein solubilizeshydrophobic ANSA, and then the hydrophobic phase formed by nonpolarprotein residues acts as an efficient micellar catalyst of *NO oxidationand formation of the nitrosating agent N₂O₃. Finally, if an NH₂ group ofANSA is available, the nitrosation reaction converts ANSA into thehighly reactive aryl-diazonium cation that immediately crosslinks theprotein interior. This suppresses the enzymatic activity of the protein.

[0023] According to the present invention, ANSA derivatives areadministered to inactivate proteins using the nucleophilic NH₂ group inthe ANSA as a suicide-nitrosative substrate. These compounds can be usedas antipathogen or anti-cancer agents, and the selectivity andspecificity of the compounds is determined by the peptide attached tothe amino group, R₃. Peptidyl-ANSA molecules are resistant to activationby nitrosation until they have been transported into the cell and thetarget peptide has been cleaved by a particular protease or peptidase atthe protein site. Because high concentrations of *NO are associated withhost defense systems against microbial infections and some tumor cellsas well, these molecules preferentially target those cells. Peptidetransport and protease activities are species- or tissue-specific, suchthat the precise design of peptidyl-ANSA molecules permits selectivetherapy for infections or cancerous tumors.

[0024] Because the ANSA derivatives of the present invention inactivateproteins per se, they are particularly useful as broad-spectrumantibiotics, antifungals, and antivirals. The action of the ANSAderivatives depends solely on nitrosation, and therefore their action isnot dependent upon the particular structure of the pathogen to bedestroyed.

[0025] The hydrophobic derivatives of ANSA, with a free R₃ NH₂ group,can thus be used as suicide substrates for different enzymes in thepresence of *NO and thus serve as a paradigm for the design of a newclass of selective antibiotics. Since many enzymes can be targeted bythese ANSA derivatives, the unique advantage of ANSA-based cytotoxins isthat they can be designed to combat many different types of cells orproteins which are exposed to *NO or which produce *NO endogenously.Since there is a general positive relationship between the grade ofmalignancy and the amount of NOS content in tumors (Thomsen et al, 1994;Klotz et al, 1998; Thomsen et al, 1995; Ambs et al, 1998; Cobbs et al,1995), including that of prostate tumors, ANSA will preferentiallytarget cancer cells. It has been demonstrated that cancer cells are moresensitive to hydrophobic ANSA derivatives than are non-transformed cellsin general, apparently because of higher endogenous *NO production bycancerous cells. The higher level of selectivity of ANSA compounds canbe determined by which peptide is attached at the R₃ NH₂ group.Peptidyl-ANSA can be synthesized to be resistant to activation bynitrosation until transport into the cell or absorption to the cellmembrane and the peptidyl ligand has been enzymatically removed, asshown in Figure Active peptide transport and protease activities arecell- or tissue-specific so that the precise design of peptidyl-ANSAmolecules makes it possible to selectively target a particular cancercell or a particular pathogen.

[0026]FIG. 5 shows that the peptide attached to the R₃ NH₂ groupinactivates the ANSA. Enzymatic cleavage of the peptide inside the cellor on the cell surface enables the ANSA to be activated by nitrosation,crosslink to proteins, and kill the cell.

[0027] Another method of targeting ANSA to a particular pathogen is byusing non-conventional or modified amino acid residues, such as D-aminoacids, which are cleaved in prokaryotic cells but not in eukaryoticcells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028]FIGS. 1A and 1B show micellar catalysis of *NO oxidation andnitrosation of biological substrates (e.g., proteins).

[0029]FIG. 2A shows the chemical structures and properties of5-aminonaphthaline sulfonamides (ANSA). FIG. 2B shows the mechanism ofANSA-dependent protein inactivation.

[0030]FIGS. 3A and 3B show inactivation of E. coli RNA polymerase by *NOand ANSA derivatives (Nedosapasov et al, 2000).

[0031]FIGS. 4A and 4B illustrate nitrosation of a test protein byaerobic addition of a solution of *NO.

[0032]FIG. 5 shows peptidyl-ANSA as a selective *NO⁻ induciblechemotoxin.

[0033]FIG. 6 illustrates synthesis of peptidyl-ANSA substrates.

[0034]FIG. 7A shows reaction of inactivation of biomacromolecules byaromatic amines via micellar oxidative nitrosation.

[0035]FIG. 7B shows aminonaphthalenesulfonamide (ANSA) and its peptidylderivatives.

[0036]FIG. 8 illustrates how peptidyl-ANSA acts as a NO⁻ dependentantibiotic.

[0037]FIG. 9 illustrates some ANSA derivatives.

[0038] FIGS. 10A-10D show comparative analysis of Cys nitrosation.

[0039] FIGS. 11A-11B show comparative analysis of Trp nitrosation.

[0040] FIGS. 12A-12C illustrate NSA-mediated catalysis of ANSAnitrosation.

DETAILED DESCRIPTION OF THE INVENTION

[0041] ANSA derivatives are prepared to inactivate proteins uponnitrosation with NO and O₂ and, thus, can be used as antibiotics,antifungals, and antivirals and anti-cancer agents. The ANSA derivativesare designed to be specific to desired cells by altering the peptidylsubstituents on the ANSA so that the derivative is selective to theparticular pathogen or cancer cells to be targeted. Hydrophobicsubstituents are used to ensure that the derivatives are hydrophobic andare preferentially delivered to the hydrophobic site of a protein.

[0042] As shown in FIG. 2B, agent (N₂O₃) first a protein solubilizes thehydrophobic ANSA. Then the hydrophobic phase formed by nonpolar proteinresidues acts as an efficient micellar catalyst of *NO oxidation andformation of the nitrosating agent. Finally, if an amino group of ANSAis free, the nitrosation reaction converts the amino group into a highlyreactive aryldiazonium cation that immediately cross-links to theprotein interior.

[0043] The hydrophobic compartments of proteins concentrate *NO andoxygen and, thus, catalyze formation of dinitrogen trioxide, N₂O₃, theprimary nitrosating agent, as shown in FIG. 2B.

[0044] Experimental Procedures

[0045] *NO/H₂O or *NO/DMSO solutions were prepared in the airtightdevice by bubbling *NO gas (Aldrich) that had been purified from higheroxides by passing it through a 1 M solution of KOH, into water or DMSO(Aldrich) until the concentration of dissolved *NO reached 1.2 mM. Water(Milli-Q grade) was deaerated by boiling and then cooling under argon(Praxair). The *NO concentration immediately before the reaction wasmeasured by using ISO—NO Mark II nitric oxide electrode (WPI, Inc). DMSOwas dehydrated by distillation in vacuum over CaO.

[0046] BSA, CM-BSA, GSH, Trp, Cys (Sigma) and the Trp-peptide (USBiological) were dissolved in water (1 mM stock solutions). Thenitrosation reaction was carried out at room temperature in the 1 mlquartz cuvette. The blank probe contained 0.5 ml K₂HPO₄/KH₂PO₄ buffer(25 mM) pH=7.0 and 0.4 ml each of the tested reagent. 0.4 ml of anaqueous *NO solution was added for 5, 10, 15, 20, 25, or 30 minutes,then 0.1 ml of 0.1% ammonium sulfamate in 0.4N HCL was added for 1minute to remove HNO₂ from the sample and UV-Vis spectra was recorded byusing Ultrospec 3000 spectrophotometer (Pharmacia). Spectra weredigitized and analyzed by Win DIG and Origin 6.0 software.

[0047] ANSA were synthesized from 5-nitronaphthalenesulpho-acids and5-aminonaphthalenesulphoacids (Reahim). TLC of ANSA and theirderivatives was carried out on Silica gel 60 plates (Merck) in thehexane/ethyl acetate system. Gel filtration was performed using theSephadex G25M size-exclusion column (Pharmacia). Fluorescence of ANSAwas observed and measured under 365 nm UV light using BioRad Fluor-SMultiImager system.

[0048] Transcription reactions (total volume=20 μl) were performed in abuffer containing 20 mM Tris HCL, pH 7.5, 10 mM MgCl₂, 50 mM KCl, 8 μMATP, GTP, UTP, 1.8 μM CTP, and 0,15 μM [α-³²P] CTP (3000 Ci/mmol) for 5minutes at 37° C. RNAP was purified as described (Nudler et al, 1996).The DNA template containing the A1 promoter of phage T7 was obtained bypolymerase chain reaction from the plasmid pENtR2 (Nudler et al, 1996).

[0049]FIG. 6 illustrates synthesis of peptidyl-ANSA substrates bysolid-phase peptide synthesis using standard 9-fluorenylmethoxylcarbonyl(Fmoc) protocols. In FIG. 6, the circles stand for Rink Amide resin(Novabiochem), DMF is dimethylformamide, HATU isO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, DICI is diisoproplycarbodiimde, HOBt is1-hydroxybenzotriazole, TFA is trifluoroacetic acid, and TIS istriisopropylsilane.

[0050] Protein Hydrophobic Core Is a Catalyst of Nitrosylation. Theobserved phenomenon of BSA-mediated catalysis of its own nitrosylationcan be explained by the principle of micellar catalysis: the hydrophobiccore of BSA accelerates formation of N₂O₃ by concentrating *NO and O₂from water solution. To test this hypothesis directly the presentinventors synthesized fluorescent versions of Griess indicators ofnitrosation, 5-aminonaphthalene-sulfonamides (ANSA). ANSA are aromaticamines that contain a sulfonamide group with one or two aliphaticsubstitutions (R₁ and R₂) nitrosylation (Nedosapasov et al, 2000) (FIG.12A). ANSA has a relatively low fluorescence yield in polar solvents,which is greatly enhanced in the nonpolar environment. Differences inthe fluorescence activity can be readily visualized by the naked eye.Nitrosation of the NH₂ group converts the ANSA molecule to anaryldiazonium cation, which cross-links with another ANSA molecule(azocoupling reaction) to generate non-fluorescent deeply coloredazoderivatives (FIG. 12A). The origin of each azoderivative dye can bedefined by thin-layer-chromatography (TLC). Additionally, thehydrophobicity of ANSA can be changed by varying the identity of R₁ andR₂ groups.

[0051] The present inventors first synthesized two ANSA with oppositehydrophobic properties (FIG. 12B). In the low-hydrophobic ANSA molecule(ANSA I), the R₁ group is —CH₃ and the R₂ group is —H. The highlyhydrophobic ANSA (ANSA II) has C₃H₇ for both R₁ and RExperiments weredone by mixing an aqueous solution of ANSA with the test protein, andthen initiating the nitrosation reaction by the aerobic addition of anaqueous solution of *NO. Two initial concentrations of *NO were used at25 molar excess over ANSA (high dose) and a 5 molar excess (low dose).In the absence of BSA, treatment of the ANSA I+ANSA II mixture with thehigh dose of *NO almost completely extinguished the fluorescence FIG. 4Aat the same rate (FIG. 4B). It produced a set of dyes that accumulatedproportionally and originated from both ANSA I and ANSA II (FIG. 12C,lane 4) indicating that both types of ANSA were nitrosated at the samerate. Under the same conditions, the low dose of *NO had littlenitrosating effect since the fluorescence was decreased only slightly(FIG. 12B, lane 6). No nitrosation of ANSA under oxygen-free, argonconditions was observed, even when the high dose of *NO was applied(data not shown), indicating that the nitrosating agents must beintermediates of *NO oxidation. Since the pH of the reaction buffer was7.5, it is likely that N₂O₃ rather then HNO₂ was the major nitrosatingagent.

[0052] In the presence of BSA, the results of nitrosation werecompletely different (FIG. 4A). ANSA II, but not ANSA I, was able toform a stable complex with BSA as evidenced by gel filtration analysis(not shown). Addition of BSA to the mixture of ANSA I+ANSA II increasedthe fluorescence of the solution significantly, (FIG. 12B, lane 2)indicating that ANSA II was solubilized by the hydrophobic core of BSA.The low dose of *NO was enough to decrease the fluorescence of the ANSAI+ANSA II+BSA mixture to approximately half that of the intact ANSAI+ANSA II mixture (FIG. 4A, compare lanes 3 and 5), indicating thatapproximately half the ANSA molecules became nitrosated. Since no dyeformation with BSA and the low dose of *NO was observed (FIG. 12C, lane7), it was concluded that only BSA-bound ANSA II, not free ANSA I, wasnitrosated. This conclusion was confirmed by isolating intact ANSA Ifrom the mixture by gel filtration and CHCl₃ extraction. The absence ofANSA II-generated dyes can be explained only if azoderivatives of ANSAII were cross-linked to amino acid residues of BSA and not to other ANSAmolecules. Correspondingly, when the high dose of *NO was applied onlydyes that originated from ANSA I were observed (FIG. 12C, lane 8). Theseresults indicate that BSA acts as a catalyst of nitrosation of the boundhydrophobic compounds (ANSA II), i.e., the nitrosating activity appearsto be greater in the hydrophobic interior of BSA then in solution.

[0053] The important conclusion from these observations is that varioussoluble proteins provide the environment for effective nitrosation ofnot only their own nucleophiles, but also of external molecules such asANSA. Thus, the hydrophobic phase formed by plasma protein serves as amajor reservoir of NO and its reactive oxides and plays an importantrole in maintaining the pool of RS-NO in vivo.

[0054] Hydrophobic Amines (ANSA) as *NO-Dependent Enzyme Inhibitors. Toinvestigate whether this mechanism might be functioning in proteinsother than BSA, the effect of nitrosation on a completely distinctprotein—RNA polymerase from E. coli (RNAP)—was examined. The experimentswere done by exposing RNAP to a low dose of *NO, and then adding the DNAtemplate and ribonucleotide substrates to initiate the transcriptionreaction. For a control, a solution of NO₂ ⁻ for the *NO was substitutedby oxidizing *NO prior to the experiment. As FIG. 3 shows, *NO partiallyinhibited transcription (lane 2) while NO₂ ⁻ had no effect (lane 3). The*NO-dependent inhibition of RNAP was complex. Nitrosation of the RNAPdecreased the overall amount of transcripts, suggesting that the earlysteps of transcription cycle were suppressed (e.g., DNA binding and openpromoter complex formation). Additionally, the addition of *NO decreasedsignificantly the accumulation of the full-length, 49 nt. transcript.Shorter transcripts represent paused RNAP molecules resulting from thelimited amount of CTP in the reaction. This redistribution of RNAtranscripts upon nitrosation suggests that the catalytic parameters ofRNAP (K_(m) for ribonucleotides and/or V_(max)) have been changed. Thecomplex inhibitory effect of nitrosation on RNAP is not surprising sincemany potential nitrosation targets are distributed in structurally andfunctionally distinct regions of the enzyme (Zhang et al, 1999).

[0055] The experiments with BSA demonstrated that upon nitrosation, ANSAII could cross-link to its “host” protein molecule. To determine whetherthis process could also occur with RNAP, the mixture of RNAP+ANSA II wastreated with *NO (FIG. 3, lane 4). In the presence of ANSA II, the samelow dose of *NO as used in lane 2 completely inactivated RNAP. ANSA IIalone did not affect transcription (lane 5).

[0056] To further test the autocatalytic mechanism of enzymeinactivation and the key role of nitrosation-azocoupling in this processadditional ANSA was synthesized and were tested in the *NO-dependentRNAP inactivation assay (FIG. 3). FIG. 3B shows ANSA that were designedto have different degrees of hydrophobicity (I<III≈IV<VI<V≈VII<II). Itwas found that the hydrophobicity of ANSA directly correlated with itsability to stimulate the *NO-dependent inactivation of the enzyme. Theleast hydrophobic ANSA I had no effect on transcription (lane 10), ANSAIII and ANSA IV exhibited a partial negative effect (lanes 6 and 14),and ANSA V completely inactivated RNAP (lane 12). ANSA VI and ANSA VII,with methyl and peptidyl substitutions in the NH₂ group, were incapableof forming aryldiazonium cation upon nitrosation. Although ANSA VI andANSA VII were more hydrophobic then ANSA III and ANSA IV, they wereunable to enhance inactivation of RNAP (lanes 8 and 16). This experimentraises the possibility that hydrophobic amines could be used asefficient tools for the *NO-dependent inactivation of enzymes.

[0057] The results with ANSA can be described by the following scheme(FIG. 2B). First, a protein solubilizes hydrophobic ANSA. Then, thehydrophobic phase formed by nonpolar protein residues acts as anefficient micellar catalyst of *NO oxidation and formation of thenitrosating agent. Finally, if an NH₂ group of ANSA is free, thenitrosation reaction converts it into highly the reactive aryldiazoniumcation that immediately cross-links to the protein interior andsuppresses the enzymatic activity.

[0058] The data obtained above suggest a mechanism in which hydrophobiccompartments of proteins concentrate *NO and O₂ and, thus, catalyzeformation of N₂O₃, the primary nitrosating agent (FIG. 1). As soon asN₂O₃ is formed within the protein interior, it attacks nearbynucleophilic amino acid groups. Short-lived intermediates of thisreaction transfer the nitroso-group further to less nucleophiliccompetitors and finally to molecules of water or thiols in thesurrounding media. This model, by which nitrosating agents are createdin regions of hydrophobicity, accounts for the high selectivity ofS-nitrosylation protein modifications (Stamler et al, 1998). In thiscase the overall structure of the protein (the size and geometry of thehydrophobic core and distribution of nucleophiles) determines itsability to generate N₂O₃ and also transfer NO⁺ to a particular Cystargets.

[0059] According to this model, N₂O₃ is synthesized not only duringnitrosative stress, i.e., when the concentration of *NO donors insolution rises substantially (Grisham et al, 1999; Espey et al, 2000;Stamler et al, 1998, and references therein), but constitutively in thehydrophobic protein interior where the local concentration of *NO and O₂is much higher then in solution. Thus, the concentration of N₂O₃ dependsnot only on the initial concentrations of *NO and O₂ in the wholesystem, but also on the size and geometry of the hydrophobic phase, aswell as the distribution of available targets in the protein molecule.Such a mechanism further suggests that both S-nitrosylation andN-nitrosation can be controlled not only by the activity of NO-synthasesand O₂ concentration but also by conformational transitions in theprotein molecule that change its hydrophobic properties. Additionally,nitros(yl)ation itself may be able to induce further conformationaltransitions, which may be favorable or unfavorable to continue theprocess. Further, the solubilization of hydrocarbons and otherhydrophobic compounds by proteins could affect nitrosation due to theincreased efficiency of micellar catalysis.

[0060] In vivo, protein-mediated catalysis of *NO oxidation is likely tocompete with a similar process within lipid membranes (Goss et al,1999). It is remained to be determined which reaction and under whatconditions contributes most to the formation of primary nitrosativespecies at the cellular and subcellular levels.

[0061] As is shown here, BSA and RNAP provide the environment foreffective nitrosation of not only their own nucleophiles but alsoexternal molecules such as ANSA. In a separate study (R. Rafikov, OlgaRafikova, and E. Nudler, manuscript in preparation), it demonstratedthat albumin significantly stimulates formation of vasoactivelow-molecular-weight nitrosothiols via the mechanism of micellarcatalysis. Q_(NO) for BSA/H₂O was determined to be ≈2. Taken together,these data suggest that the hydrophobic phase formed by plasma proteinserves as a major reservoir of *NO and its reactive species and plays animportant role in maintaining the pool of RS—NO in vivo.

[0062] Here it is demonstrated that the ability of a protein toaccelerate nitrosation can be directed towards its own inactivation.ANSA molecules with the nucleophilic NH₂ group can be used as a suicidenitrosative substrate for different proteins and, thus, can serve as aparadigm for the design of a new class of antibiotics. The highselectivity and specificity of such compounds can be determined by thepeptide attached to the NH₂ group (FIG. 8). Peptidyl-ANSA molecules canbe synthesized which are resistant to activation by nitrosation untilthey have been transported into the cell and the peptide has beencleaved by a particular protease or peptidase. Since high concentrationsof *NO are associated with host defense systems against microbialinfections and also some tumor cells, such molecules wouldpreferentially target those cells. Peptide transport and proteaseactivities are species- or tissue-specific so that the precise design ofpeptidyl-ANSA molecules allows the exciting possibility for new kinds ofselective therapy. One skilled in the art can, without undueexperimentation, readily design peptidyl-ANSA molecules that can targeta desired protein so as to target specific bacteria, viruses, fungi,cancer cells, etc.

[0063] ANSA Derivatives as a New Class of Antibiotics and AntineoplasticDrugs. ANSA derivatives (shown in FIG. 9) were tested for theircytotoxic capabilities using wild type strains of Gram-negative andGram-positive bacterial (Escherichia coil strain BL21 and Bacillussubtilis Strain IS75). As Table 1 shows, hydrophobic ANSA exhibitedstrong bactericidal effects, which directly correlated with the amountof NO produced by different bacteria under various growth conditions. NOtrap, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO),almost completely abolished the inhibitory effect of ANSA on the amountof colony forming units (CFU), while addition of a water solution of NOrestored the bactericidal effect of ANSA, thus indicating that thetoxicity of ANSA was strictly dependent upon nitrosation. Aliphatic R₁and R₂ substitutions, as well as the R₃ NH₂ group, were essential forgrowth inhibition. ANSA VIII that contained a non-cleavable R₃CH₃ had noeffect on cell survival. Notably, ANSA with a peptidyl R₃ substitution(ANSA VII) exhibited the strongest bactericidal effect. TABLE 1Bacterial Properties of Different ANSA Derivatives E. coli B. subtilisrich minimal rich minimal media — ≈10⁷ ≈10⁷ ≈10⁷ ≈10⁷ CFU ml⁻¹ *NO ≈10⁶≈10⁶ ≈10⁶ ≈10⁶ ANSA 1 10⁶-10⁷ ≈10⁷ ≈10⁷ ≈10⁷ ANSA V  0-20 ≈10³ 10²-10³10² ANSA VII 0 ≈ ANSA VIII ≈10⁷ ≈10⁷ ≈10⁷ ≈10⁷ ANSA V + *NO 0 0 0 0 ANSAV + PTIO ≈10⁵ ≈10⁵ 10⁵-10⁶ ≈10⁵ ANSA V + *NO +  5-10 0.05-0.2 1-2 ≈2[NO] PTIO μM #ANSA VIII is the same as ANSA V but with a CH₃.[*NO] wasmeasured directly in growing cultures by using a Clark-typeNO-electrode.

[0064] A second preliminary study used two human hormone-independentprostatic carcinoma cell lines DU-145 and PC-3 (ATCC, Virginia) andnormal human fibroblasts (WI-38). The MTS cytotoxicity assay (“CellTiter96”, Promega) was used to measure the cytotoxicity of several ANSAcompounds in the presence or absence of *NO. All compounds tested invitro were dissolved in DMSO (100 μM solution) and subsequently dilutedin the culture medium before treatment of the cultured cells. Testedcells were plated in 96-well plates at a density of 4×10³ cells/well/200μL of the proper culture medium and treated with the compounds atconcentrations of 0.1-100 μM. In parallel, the cells were treated with1% of DMSO as control. MTS assay was performed 72 h later according toinstructions provided by the manufacturer (Promega). This assay is basedon the cellular conversion of the tetrazolium salt,MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt], into a formazan that is soluble in cell culture medium andis measured at 490 nm directly in 96-well assay plates withoutadditional processing. Absorbency is directly proportional to the numberof living cells in culture.

[0065] Hydrophobic ANSA, but not hydrophilic ANSA I, exhibited a strongcytotoxic effect, which directly correlated with the amount of exogenous*NO provided. In one control, the NO trap,2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), was used,which decreased the inhibitory effect of *NO in the presence of ANSA,thus indicating that the toxicity of ANSA strictly depended onnitrosation. Since, the general NOS inhibitor, L-NAME, also decreasedthe cytotoxicity of ANSA it was concluded that endogenous production of*NO in prostate cancer cells at least partially is responsible forintrinsic ANSA cytotoxicity. Aliphatic R₁ and R₂ substitutions as wellas the R₃ NH₂ groups were essential for cell growth inhibition. ANSAVIII, which is the same as ANSA V but contained a non-cleavable R₃ CH₃,had no effect on cell survival. Notably, ANSA with a peptidyl R₃substitution (ANSA VII) (FIG. 9) exhibited the strong cytotoxic effect.In order to be nitrosated, peptidyl-ANSA must undergo cleavage of theC-terminal amino acid residue to relieve the R₃ NH₂ group. Takingadvantage of the chromogenic nature of ANSA compounds we were able todetermine whether the peptidyl-ANSA were transported in the cell and/orbound to the cell membrane and processed by proteolysis. Duringincubation with ANSA VII cultured cells turns yellowish. After celldisruption followed and sedimentation much of yellow stain remained inthe supernatant. Yellow dye formation is indicative of the azocouplingreaction between ANSA molecules (FIG. 2A) suggesting that the peptidylgroup indeed had been removed and that nitrosation occurredintracellular or in the membrane.

[0066] In order to be nitrosated, peptidyl-ANSA must undergo cleavage ofthe C-terminal amino acid residue to relieve the R₃ NH₂ group. Takingadvantage of the chromogenic nature of ANSA compounds, it was possibleto determine whether the peptidyl-ANSA were transported in the cell andprocessed by proteolysis. During incubation with ANSA VII, E. coli cellsturned deeply yellow. After mild cell disruption followed bysedimentation of the membrane debris, much of the yellow stain remainedin the supernatant. Yellow dye formation is indicative of theazocoupling reaction between ANSA molecules, suggesting that thepeptidyl group had indeed been removed and that nitrosation occurredintracellularly. Unlike most common bacteriotoxic antibiotics, ANSAderivatives kill bacterial cells extremely rapidly. Minimal inhibitoryconcentrations of ANSA derivatives were comparable with that of moststandard antibiotics.

[0067] ANSA derivatives also can be used to kill viruses, fungi, andother pathogens extremely rapidly by inactivation of these proteins.Minimal inhibitory concentrations of the ANSA derivatives can be used asantifungals, antivirals, etc. because of the rapid action of thesecompounds.

[0068] Thus, the ANSA derivatives of the present invention are designedto target proteins and suppress enzymatic activities of these proteinsupon nitrosation. While these compounds are particularly useful asbactericides and anti-tumor compounds, they also are effective againstany other types of biomacromolecules that are undesirable, including butnot limited to viruses, enzymes, toxins, and the like. The peptidylmoiety can be designed to recognize a specific protein receptor, and thehydrophobic groups help directed the ANSA derivative to the appropriatelocation in the protein.

[0069] The ANSA derivatives of the present invention are hydrophobicderivatives of 5-aminonaphthalenesulfonamides with a free amino groupwhich is coupled to a peptidyl group. These compounds are used as asuicide nitrosative substrate for different enzymes, including bacterialRNA polymerase and beta-galactosidase and, thus, can be used as a newclass of antibiotics. Since virtually any enzyme can be targeted by theANSA derivatives, the unique advantage of using the ANSA derivatives ofthe present invention is that they can be designed to combat diversetypes of cells, including bacteria, and cancer cells as well as diversetypes of viruses.

[0070] Many infectious bacteria constitutively produce substantialamounts of NO via enzymatic reduction of nitrite/nitrate. On the otherhand, there is a general positive relationship between the grade ofmalignancy and the amount of NOS in tumors. Furthermore, the high levelof inducible NO is generally associated with host defense againstmicrobial infections as well as cancer cells, and ANSA derivatives canbe designed to target those cells specifically.

[0071] The most common infectious bacterial species, includingStaphylococcus spp., Escherichia coli, Mycobacterium, and helicobacterpylori, are facultative anaerobes that use nitrate/nitrite besidesoxygen as an electron acceptor or oxidation of carbon compounds toderive energy under microaerobic conditions. NO is an intermediate inthe process of denitrification formed by the action of the respiratorynitrite reductases cytochrome cd1 or copper nitrite reductase.

[0072] Tumor cells are usually characterized by increased intracellularand extracellular levels of NO produced by their own NOS as well as iNOSof activated tumor invading macrophages. They are also characterized byhyperactive extracellular proteolysis. In particular, the extracellularmatrix metalloproteinases and the serine proteinase, urokinase-typeplasminogen activator, are most extensively linked to cancer invasionsand metastasis. Superposition of two selective factors, high localconcentration of NO and intensive proteolysis, provides an opportunityfor ANSA-based chemotherapy. However, unlike the situation withbacteria, in order to be activated peptidyl-ANSA need not necessarily betransported inside the cell, but rather inserted into the membrane viaR₁/R₂ aliphatic tails. The exposed peptidyl substrate is then availablefor extracellular proteolysis.

[0073] To construct selective peptidyl variations of ANSA, a nonrandomapproach includes incorporation of peptidyl groups that have beenpreviously selected as specific substrates for the metastasis associatedproteases. For example, a specific substrate for uPA,NH₂-Asp-Thr-Ala-Arg-X, has been selected from the synthetic library of137,180 substrate members. Another peptide, NH₂-Gly-Pro-Leu-Gly-/X, wasselected from a phage display library containing 2×10⁸ independentrecombinants as a specific substrate for MMP-13. These and otherspecific peptidyl derivatives of ANSA were synthesized according topublished protocols.

[0074] A random combinatorial approach involves synthesis of apositional scanning library of fluorogenic peptidyl-ANSA to define thesubstrate specificity or different cancerous cell lines. In contrast toother combinatorial libraries, this library format provides rapid andcontinuous information on each of the varied substituents in thesubstrate. A positional scanning library is prepared so that eachsubstrate occupies its own known spot in the array. Specific cleavage ofthe amide bond after the X residue liberates the fluorescent ANSA, thusallowing for a simple determination of cleavage rates for a library ofsubstrates.

[0075] Library synthesis is performed by attaching Fmos-protected ANSAto a solid support through its sulfonyl group. Fmoc solid phase peptidesynthesis is performed according to standard protocols in 96-well platesusing the FlexChem organic synthesis system of Robbins Scientific.Support-bound peptidyl-ANSA is released in solution, dissolved in DMSO,and transferred to 96-well Microfluor plates for fluorescence analysis.Nineteen natural amino acids (excluding Cys and Met) and unnaturalproteinogenic amino acids can be readily incorporated at each of fourpositions, given more than 13,300 individually localized substrates. Thecompleted library is used for throughput substrate screening forindividual proteolytic enzymes, as well as proteolysis by whole cells.

[0076] The ANSA derivatives to be used to target proteins according tothe present invention are those in which both R₁ and R₂ are hydrophobicgroups, and R₃ is preferably a peptidyl group. While not limited to anyparticular hydrophobic groups, as the type of hydrophobic groups andpeptidyl group will depend upon the particular protein targeted, thehydrophobic groups are preferably selected from groups having from about4 to about 30 carbon atoms. Among the hydrophobic groups that can beused are hydrocarbon groups of 10-16 carbon atoms, which can bebranched, cyclic, or linear, with varying degrees of saturation andsubstitution, carbobenzoxyl, dansyl, vinyl, phenyl, tolyl, and the like,substituted with substituents such as epoxy, mercapto, polyamino, andalkoxycarbonyl.

[0077] Preferably, the ANSA derivatives have the following formulae:

[0078] wherein R₁, R₂ and R₄ are C₁-C₈ alkyl, alkyl halide, alkenyl,alkenyl halide, aryl, arylalkyl, and aryl and arylalkyl halides or anaffinity group and at least one of R₁, R₂ and R₄ is a hydrophobic group;and R₃ is a peptide group.

[0079] The ANSA derivatives of the present invention can be used totarget pathogenic bacteria. As multiple antibiotic-resistant strains ofpathogenic Gram-positive and Gram-negative bacteria have emerged, publichealth has been seriously threatened because of lack of means to controlthese bacteria. For example, the ANSA derivatives of the presentinvention can be designed to target bacteria that cause wound andbloodstream infections, such as vancomycin-resistant Staphylococcusaureus or sepsis and meningitis caused by multiple antibiotic-resistantStreptococcus pneumoniae strains. These infections are much more commonand threatening for hospitalized and/or otherwise exhausted individualsin which standard host defense mechanisms are compromised. Thus, theANSA derivatives of the present invention can be used as a new class ofbactericidal drugs.

[0080] The three groups (R₁, R₂ and R₃) chemically attached to ANSA cancreate virtually an unlimited diversity, and thus these compounds can bemade highly specific for their targets. This specificity can be used forrecognition and inactivation of only particular types of proteins (e.g.,regulatory transcription factors or receptors) as well as for wholecells, such as bacterial cells or cancer cells.

[0081] Because the ANSA derivatives are not effective until they aretransported to the target cells and enzymatically converted to ANSA inthe hydrophobic portion of a cell, they are relatively harmless to cellsthat are not targeted. The permeability and cleavage of the ANSAderivatives are determined by the structure of the peptide and the ANSA.

[0082] ANSA derivatives according to the present invention can beadministered by any convenient route, including parenteral,subcutaneous, intravenous, intramuscular, intra peritoneal, ortransdermal. Alternatively or concomitantly, administration may be bythe oral route. The dosage administered depends upon the age, health,and weight of the recipient, nature of concurrent treatment, if any, andthe nature of the effect desired.

[0083] Compositions within the scope of the present invention includeall compositions wherein the active ingredient is contained in an amounteffective to achieve its intended purpose, notably, inhibition ofinfection or cancer cells. While individual needs vary, determination ofoptimal ranges of effect amounts of the ANSA derivatives is within theskill of the art. Typical dosages comprise from about 10 nanograms/kg toabout 100 mg/kg of body weight.

[0084] Pharmaceutical compositions for administering the ANSAderivatives of the present invention preferably contain, in addition tothe ANSA derivative, suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Preferably, the preparations, particularly those preparations which areadministered orally, and which can be used for the preferred type ofadministration, such as tablets, dragees, and capsules, as well assuitable suspensions for administration by injection or orally,containing from about 0.0001 to about 99 percent by weight of activecompound, together with the excipient. For purposes of the presentinvention, all percentages are by weight unless otherwise indicated. Inaddition to the pharmaceutical compositions described here, thecompounds of the present invention can be formulated as inclusioncomplexes, such as cyclodextrin inclusion complexes.

[0085] The pharmaceutically acceptable carriers include vehicles,adjuvants, excipients, or dilutes that are well known to those skilledin the art and which are readily available. It is preferred that thepharmaceutically acceptable carrier be one which is chemically inert tothe active compounds and which has no detrimental side effects ortoxicity under the conditions of use.

[0086] The choice of carrier is determined partly by the particular ANSAderivative, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of the pharmaceutical compositions of the presentinvention. Formulation can be prepared for oral, aerosol parenteral,subcutaneous, intravenous intraarterial, intramuscular, intraperitoneal,intratracheal, rectal, and vaginal administration.

[0087] Suitable excipients are, in particular, filers, such assaccharides (lactose, sucrose, mannitol, sorbitol), cellulosepreparations, and calcium phosphates, such as tricalcium phosphate orcalcium hydrogen phosphate. Binders include starch paste using maizestarch, wheat starch, rice starch, potato starch, or the like, gelatin,tragacanth, methyl cellulose, hydroxypropylmethylcellulose, and/orpolyvinylpyrrolidone.

[0088] Other pharmaceutical carriers include liposomes. Since the ANSAderivatives are hydrophobic, the active ingredient is present in thelipidic layer. The lipidic layer generally, but not exclusively,comprises phospholipids such as lecithin and sphingomyelin, steroidssuch as cholesterol, more or less ionic surfactants such as dicetylphosphate, stearylamine, or phosphatidic acid.

[0089] The compounds may be formulated for transdermal administration,such as in the form of transdermal patches, so as to achieve systemicadministration.

[0090] Parenteral formulations can be prepared using oils such aspetroleum, animal, vegetable, or synthetic oils. Alternative carriersare fatty acids, such as oleic acid, steric acid and isostearic acid.

[0091] In determining dosages of the ANSA to be determined, the dosageand frequency of administration is selected in relation to thepharmacological properties of the specific active ingredients. Intoxicity studies in general, the highest dose should reach a toxic levelbut be sublethal for most animals in the group. The lowest dose shouldinduce a biologically demonstrable effect.

[0092] The amount of ANSA derivatives to be administered to any givenpatient must be determined empirically, and will differ depending uponthe condition of the patient. Relatively small amounts of the activeingredient can be administered at first, with steadily increasingdosages if no adverse effects are noted. Of course, the maximum safetoxicity dosage as determined in routine animal toxicity test shouldnever be exceeded.

[0093] The foregoing description of the specific embodiments will sofully reveal the general nature of the invention that others can, byapplying current knowledge, readily modify and/or adapt for variousapplications such specific embodiments without undue experimentation andwithout departing from the generic concept. Therefore, such adaptationsand modifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means andmaterials for carrying our various disclosed functions make take avariety of alternative forms without departing from the invention. Thus,the expressions “means to . . . ” and “means for . . . ” as may be foundin the specification above and/or in the claims below, followed by afunctional statement, are intended to define and cover whateverstructural, physical, chemical, or electrical element or structureswhich may now or in the future exist for carrying out the recitedfunction, whether or not precisely equivalent to the embodiment orembodiments disclosed in the specification above; and it is intendedthat such expressions be given their broadest interpretation.

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What is claimed is:
 1. A method for inactivating biomacromolecules byadministering an effective amount of an ANSA derivative of the formula:

wherein at least one of R₁ and R₂ are selected from the group consistingof hydrophobic groups and affinity groups, with the proviso that atleast one of R₁ and R₂ is a hydrophobic group, and R₃ is selected fromthe group consisting of amino acyl groups and peptidyl groups:
 2. Themethod according to claim 1 wherein the biomacromolecules are bacteria.3. The method accordant to claim 1 wherein the biomacromolecules arefungi.
 4. The method according to claim 1 wherein the biomacromoleculesare viruses.
 5. The method according to claim 1, wherein thebiomacromolecules are enzymes.
 6. The method according to claim 6,wherein the enzymes are bacterial enzymes.
 7. The method according toclaim 1, wherein the biomacromolecules are regulatory transcriptionfactors or receptors.
 8. The method according to claim 1 wherein thebiomacromolecules are cancer cells.
 9. A method for inactivatingbiomacromolecules by administering an effective amount of an ANSAderivative of the formula:

wherein R₁, R₂ and R₄ are selected from the group consisting of C₁-C₃₀alkyl, alkyl halide, alkenyl, alkenyl halide, aryl, arylalkyl, and aryland arylalkyl halides and at least one of R₁, R₂ or R₄ is a hydrophobicgroup; and R₃ is an amino acyl or a peptide group.
 10. Derivatives ofANSA of the following formula:

wherein R₁ and R₂ are selected from the group consisting of hydrophobicgroups and affinity groups with the proviso that at least one of R₁ andR₂ is a hydrophobic group, and R₃ is selected from the group consistingof amino acyl groups and peptidyl groups.
 11. Pharmaceuticalcompositions for inactivating proteins comprising an effective amount ofa compound according to claim 10 and a pharmaceutically acceptablecarrier.
 12. Derivatives of ANSA of the following formulae:

wherein G is glycine and R is arginine.
 13. Pharmaceutical compositionsfor inactivating proteins comprising an effective amount of a compoundaccording to claim 12 and pharmaceutically acceptable carrier.